1. Field of the Invention
The present invention relates to a technique useful in the microbial industry. More specifically, the present invention relates to a method for producing L-lysine by fermentation.
2. Brief Description of the Related Art
L-Lysine is produced by fermentation using microorganisms that belong to the genus Corynebacterium, Bacillus, Escherichia or the like (see “Amino Acid Fermentation”, Ed. By H. Aida et al., the Japan Scientific Societies Press [Gakkai Shuppan Center], 1st Edition, published on May 30, 1986). Bacterial strains isolated from nature or mutant strains thereof auxotrophic in nutrients have been used to improve the production in these microorganisms. Furthermore, various techniques have been disclosed for increasing the L-lysine-producing ability using recombinant DNA techniques to enhance L-lysine biosynthetic enzymes (WO95/16042).
Productivity of L-lysine has been considerably increased by breeding of microorganisms such as those mentioned above as well as improvements of production methods. However, in order to respond to the increase in future demands, the development of a method which provides more efficient production of L-lysine at a lower cost is clearly necessary, and therefore, still represents a need in the art.
Methanol is a fermentation raw material which is available in large amounts at a low cost. Methods for producing L-amino acids by fermentation using methanol are known, and include methods using microorganisms that belong to the genus Achromobacter or Pseudomonas (Japanese Patent Laid-open (Kokai) No. 45-25273), Protaminobacter (Japanese Patent Publication (Kokoku) No. 49-125590), Protaminobacter or Methanomonas (Japanese Patent Laid-open (Kokai) No. 50-25790), Microcyclus (Japanese Patent Laid-open (Kokai) No. 52-18886), Methylobacillus (Japanese Patent Laid-open (Kokai) No. 4-91793), Bacillus (Japanese Patent Laid-open (Kokai) No. 3-505284), Methylophilus (WO00/61723) and so forth.
Furthermore, for strict methanol-utilizing bacteria, especially Methylophilus bacteria, it has been reported that it is difficult to obtain auxotrophic mutants by the usual methods ((1983), vol. 129, pp. 785–799; M. L. O'Connor and R. S. Hanson, Journal of General Microbiology (1978), vol. 104, pp. 105–111). Therefore, when attempts were made to obtain a glutamine auxotrophic strain, for example, only a strain having temperature-sensitive auxotrophy could be obtained (Windass J. D. et al., Nature, 287, pp. 396–401 (1980)). Even when a special procedure, for example, suspending cells of a bacterial strain in a solution containing a mutagenesis agent for DNA and applying a voltage to the cells to forcibly make holes in the cell membranes and thereby flow the mutagenesis agent into the cells (electroporation) was used, only three kinds of mutant strains, i.e., a folic acid auxotrophic strain, a strain polyauxotrophic in serine and alanine, and a strain polyauxotrophic in glutamic acid and inositol, could be obtained (C. S. Kim and T. K. Wood, Applied Microbiol. & Biotechnology, 48, pp. 105–108 (1997)).
In addition, WO00/61723 also described that Methylophilus methylotrophus was subjected to mutagenesis treatment using a chemical mutagenesis agent to obtain a leaky casamino acid auxotrophic strain, and that strain produced valine, leucine and isoleucine. However, judging from the characteristics of the mutant strain, it appears that the strain became a leaky casamino acid auxotrophic strain because the change in cell membranes allowed various amino acids in the medium to permeate the cells.